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In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Koch, Daniel J. and Chen, Mike M. and van Beilen, Jan B. and Arnold, Frances H. (2009) In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6. Applied and Environmental Microbiology, 75 (2). pp. 337-344. ISSN 0099-2240. PMCID PMC2620723. https://resolver.caltech.edu/CaltechAUTHORS:KOCaem09

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Abstract

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47{Delta}B) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1128/AEM.01758-08DOIArticle
http://aem.asm.org/cgi/content/abstract/75/2/337PublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620723/PubMed CentralArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2009, American Society for Microbiology. Received 30 July 2008/ Accepted 8 November 2008. Published ahead of print on 14 November 2008. We thank B. Witholt, A. Schmid, E. Funhoff, and M. Camps for providing materials or instructions. D.J. Koch acknowledges grant KO 3503/1-1 from the Deutsche Forschungsgemeinschaft. This study was funded by Department of Energy award DE-FG02-06ER15762. Supplemental material for this article may be found at http://aem.asm.org/.
Funders:
Funding AgencyGrant Number
Deutsche Forschungsgemeinschaft (DFG)KO 3503/1-1
Department of Energy (DOE)DE-FG02-06ER15762
Issue or Number:2
PubMed Central ID:PMC2620723
Record Number:CaltechAUTHORS:KOCaem09
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:KOCaem09
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:12886
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:08 Jan 2009 23:54
Last Modified:03 Oct 2019 00:32

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